Several hydrocarbon-producing pathways in living systems can produce molecules that are similar to those found in petrol, diesel and jet fuel, including, isoprenoids, fatty acids and polyketides. In addition to these hydrocarbons, higher alcohols (hydrocarbon chains longer than ethanol) are suitable replacements for petrol and can be used to synthesize diesel and jet fuel. Engineering microorganisms can be used to produce advanced biofuels that can directly replace petroleum-based fuels. Two major biofuels have been most widely commercialized: bioethanol and biodiesel, which are obtained from various sources.
Isoprenoids are excellent biofuel candidates, as their branched structure prevents gelling at low temperatures and increases octane number. The titres, rates and yields (TRY) of engineered bacteria and fungi to produce isoprenoids have been increased, including the development of bypass pathways to reduce ATP use.
Various approaches have been taken to improve fatty acid biosynthesis in microorganisms for biofuel production, including overexpression of acetyl-CoA carboxylase to increase malonyl-CoA levels, deletion of β-oxidation genes in the fatty acid degradation pathway, overexpression of heterologous ATP citrate lyase (which converts citrate to acetyl-CoA), circumventing the endogenous pyruvate dehydrogenase reaction to increase acetyl-CoA levels, and increasing NADPH supply by converting NADH to NADPH.
The fusel alcohols (or higher alcohols) are generally derived by catabolism of branched amino acids via the Ehrlich pathway, which is naturally found in yeast. To enhance the production of many higher alcohols, E. coli, cyanobacteria, Corynebacterium glutamicum and other bacterial hosts have been engineered to contain this pathway, by introduction of a promiscuous 2-keto acid decarboxylase and an alcohol dehydrogenase.
Figure 1 Metabolic pathways for biofuel production. (Keasling, J., et al. 2021)
Toxicity from different sources may inhibit microbial growth and production, which include components of the culture medium arising from the hydrolysed feedstock, pretreatment by-products and reagents, and altered medium conditions. Pathway intermediates, recombinant protein expression and the final product itself may also act as burden. Removal of final products or clean-up of the starting material is a powerful process strategy to address these toxicities. In addition, various mechanisms to enhance tolerance to multiple of these inhibitory factors exist, including the expression of chaperones (e.g., ameliorate protein misfolding), cell wall modification (e.g., change membrane fluidity) and coupling or uncoupling of growth from production. Model organism or highly genetically tractable microbial host has been engineered to increase tolerance. The other approach is to use microbial strains that are naturally tolerant of the inhibitory compound. These approaches have resulted in engineering of robust host chassis with enhanced growth and productivity.
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